The present invention relates generally to apparatus and methods for automated handling of objects carried in a fluid stream, in particular, to apparatus and methods for sorting living multicellular biological objects such as plant embryos.
Modern agriculture often requires the planting of large numbers of substantially identical plants selected to grow optimally in a particular locale or to possess certain other desirable traits. Production of new plants by sexual reproduction, which yields botanic seeds, is a lengthy, labor-intensive process that is often subject to genetic recombinational events resulting in variable traits in the progeny. Furthermore, inbred strains used to perform such crosses often lack vigor, resulting in low seed productivity.
Botanic seeds, such as those produced by conventional plant breeding, have food-storage organs and protective structures that shelter plant embryos from the harsh soil environment, nurture the embryo during sowing and germination, and enable the seed to survive until conditions are favorable for germination.
In view of the disadvantages of producing large numbers of identical progeny plants by sexual means, propagation of commercially valuable plants via culturing of somatic or zygotic plant embryos has been intensively studied. For some species such xe2x80x9casexualxe2x80x9d propagation has been shown to yield large numbers of genetically identical embryos, each having the capacity to develop into a normal plant. Unfortunately the resulting embryos lack the protective and nutritive structures found in natural botanic seeds. As a result, the embryos are usually cultured under laboratory conditions until they reach an autotrophic xe2x80x9cseedlingxe2x80x9d state characterized by an ability to produce their own food via photosynthesis, resist desiccation, produce roots able to penetrate soil, and fend off soil microorganisms.
Much effort has been directed to the development of techniques for embryogenesis of agronomically important plant species, including conifer species. See, e.g., U.S. Pat. Nos. 4,957,866, 5,034,326, and 5,036,007. Totipotent plant tissue is developed in culture to a stage similar to the natural zygotic embryos occurring in mature seeds. For conifers, these are very small, commonly ranging from about 2-4 mm in length. Embryos have a bipolar form which anticipates the ultimate plant. One end has a latent radicle or root, and the other end has a latent cotyledon and appears similar to a tiny crown.
Somatic embryos lack the endosperm of the natural seed. In order to provide nutrients to the embryo at the time of germination, somatic embryos may be placed on a solid germination medium that contains the necessary carbohydrate and other nutrients, on a growing medium, or on synthetic soil that is saturated with an appropriate nutrient solution. Sterility must be maintained until after the resulting plantlet is well established. Somatic embryos also lack a seed coat and thus are more susceptible than botanic seed to mechanical damage, desiccation, and attack by pathogens and pests.
A preferred method of germinating a unit of totipotent plant tissue, e.g., a plant somatic embryo, is to incorporate it into a manufactured seed (i.e., xe2x80x9cartificial seedxe2x80x9d or xe2x80x9cseed analogxe2x80x9d). A number of versions of manufactured seed have been described in the patent literature, including U.S. Pat. Nos. 4,562,663; 4,583,320; 4,615,141; 4,715,143; 4,777,762; 4,779,376; and 4,780,987 and Canadian Patent No. 1,241,552. More advanced versions of manufactured seed that display an improved germination rate are disclosed in U.S. Pat. Nos. 5,427,593 and 5,236,469, incorporated herein by reference.
Methods and apparatus are needed for producing manufactured seed on a commercial scale. If an economical production rate is to be obtained, this process must be automated as much as possible.
One step in this production of manufactured seed is the selection of totipotent plant tissue, e.g., somatic embryos, that are mature enough to incorporate into manufactured seed. There is typically significant variation in morphological normalcy and embryo maturity in somatic embryos produced by conventional tissue culture methods. Manufactured seed containing morphologically abnormal or immature embryos seldom germinate into normal plantlets. Tedious manual selection has been the standard solution to this problem.
Various apparatus have been described for sorting microscopic biological objects such as single cells. See, U.S. Pat. Nos. 3,560,754, 3,710,933, 3,791,517, 3,987,307, and 4,175,662. These apparatus are generally not useful for sorting larger, multicellular biological objects, particularly macroscopic objects such as plant embryos.
A method has been described for separating loblolly pine zygotic embryos and celery somatic embryos according to maturity criteria using sucrose density gradients (Velho et al.; HortScience, Programs and Abstracts (suppl.), p. 137, 1989 [Abstract, 87th Annual Meeting of the American Society of Horticultural Science, Tucson, Ariz., Nov. 4-8, 1990]).
U.S. Pat. No. 5,284,765 describes a method of directionally orienting plant embryos in a liquid flotation medium.
Published International Application WO 91/00781 describes the use of a scanner to identify and determine the location of plant embryos and a pipetting mechanism to remove the plant embryos from the liquid culture medium.
Harrell et al., Computers and Electronics in Agriculture 9:13-23, 1993, describes a system for classifying plant embryos. Mature embryos arc fixed, manually introduced into the system under non-sterile conditions, and optically imaged. Images of the objects are analyzed using a neural network. Objects identified as mature embryos are deflected out of a gap in a conduit in a medium-filled harvest chamber by an injection of culture medium from a control nozzle and collected. Rejected structures pass through the gap and enter a settlement chamber.
There remains a need for automated apparatus and methods for rapidly and efficiently handling multicellular biological objects such as plant embryos under aseptic conditions without subjecting the objects to mechanical forces that would cause substantial damage. In particular, there is a need for apparatus and methods for rapidly separating embryos that are acceptable for producing manufactured seed from unacceptable embryos and delivering the acceptable embryos in an aseptic fluid stream to a location for incorporation into manufactured seed.
It is an object of the present invention to provide apparatus and methods for introducing a fixed spacing between objects carried in a fluid stream.
It is another object to provide apparatus and methods for classifying objects carried in a fluid stream.
It is another object of the invention to provide apparatus and methods for sorting objects belonging to various classes.
It is another object of the invention to provide apparatus and methods for performing these functions without subjecting fragile biological objects such as plant embryos to mechanical forces, e.g., shear forces, that would damage and reduce the viability of the objects.
It is a further object of the invention to provide such apparatus and methods that maintain an aseptic environment for the biological objects to prevent contamination.
The foregoing objects have been achieved by providing apparatus and methods for directing objects carried serially by a fluid (e.g., a liquid such as water or a culture medium, air, etc.) to a desired destination. The apparatus includes an upstream, or source, fluid conduit and two or more downstream, or destination, fluid conduits. A sensor, e.g., a fiber-optic sensor, is associated with the upstream conduit and provides information regarding objects in the upstream conduit, e.g., the presence of the object at a particular location in the upstream conduit or an image of the object. A switch coupled to the upstream fluid conduit is selectively operable to deliver the fluid stream, and objects carried by the fluid stream, from the upstream conduit to the appropriate downstream conduit.
For example, according to one embodiment of the invention, the switch is selectively operable to apply a force to at least one of the upstream or downstream fluid conduits, e.g., to align an end of an upstream conduit with an end of a downstream conduit according to the information provided by the sensor to produce a single fluid-tight path for the fluid stream and for objects carried therein. Alternatively, the switch comprises a fluid chamber that is selectively operable to be aligned with an upstream conduit to receive the object and then to be moved into alignment with a downstream conduit in order to direct the object thereto.
According to one embodiment of the invention, the upstream conduit is normally connected to a first downstream conduit. A sensor associated with the upstream conduit produces a signal upon detecting the presence of an object at a particular location in the upstream conduit. The switch responds to the signal by delivering the fluid stream to a second downstream conduit. Then, after a predetermined delay, the switch reconnects the upstream conduit with the first downstream conduit. This permits the detected object and a unit volume of the fluid in which the object is carried to enter the second downstream conduit. This embodiment is useful, for example, for automatically achieving a desired spacing between objects that are randomly spaced as they enter the upstream conduit.
According to another embodiment of the invention, the apparatus includes an optical sensor associated with the upstream conduit that produces an image of an object at a particular location in the upstream conduit and transmits the image to a signal processor, which processes the image for display on a monitor for viewing and classification by a human operator, who transmits a signal corresponding with the classification of the object. Alternatively, in an automated apparatus, the signal processor transmits the processed image to a computerized image recognition system that classifies the object and produces a signal corresponding to the classification of the object that causes the switch to direct the object to the appropriate downstream conduit. Apparatus according to the invention are particularly useful for spacing and sorting living biological objects, especially fragile multicellular, macroscopic objects such as plant embryos. The apparatus are designed such that fluid flow is substantially nonturbulent (i.e., laminar) to reduce or eliminate mechanical damage to fragile objects resulting from shear forces. Moreover, the apparatus can be maintained and operated under conditions that maintain asepsis of the medium in which the objects are suspended and prevent contamination of the objects themselves.
The apparatus can be fully automated. Two or more apparatus according to the invention can be arranged in series or in parallel for spacing, orienting, or sorting objects according to multiple criteria. For example, in a commercial process for producing manufactured seed, embryos carried in a fluid stream in a randomly spaced fashion can be directed to one embodiment of the invention to achieve a regular spacing, then directed to another embodiment of the invention in series with the spacing apparatus to classify and sort the embryos and direct embryos that are acceptable for manufactured seed to a location for manufactured seed assembly.
The present invention also provides related methods for directing objects to a desired location, producing a substantially regular spacing between objects, classifying and sorting objects, and producing manufactured seed that include plant embryos sorted by such methods, as well as manufactured seed produced by such methods.
Those skilled in the art will appreciate the utility of this invention which is not limited to the specific experimental modes and materials described herein.
The foregoing and other features and advantages of the invention will become more apparent from the following detailed description and accompanying drawings.